1. Field of the Invention
The present invention relates to methods and materials for the protection of plants against pathogens through plant genetic engineering. More particularly, the invention relates to genes which enhance a plant""s ability to withstand pathogen attack by encoding proteins that physically interact with proteins encoded by disease resistance genes (R genes) in a plant""s signal transduction pathway to activate plant defense mechanisms. The invention also relates to transgenic plants and methods for making the same, the genomes of the plants having incorporated therein foreign nucleotide sequences selected in accordance with the invention which function to enhance the plants ability to resist pathogens.
2. Discussion of Related Art
Crop losses resulting from pathogenic organisms such as viruses, bacteria, fungi and nematodes is a historic and widespread problem in a wide variety of agricultural industries. These crop losses caused by pathogen-related plant damage result in economic losses amounting to billions of dollars annually. This problem has been addressed in the past by employing a wide variety of chemicals to reduce pest damage to plant crops. The approach, however, has been associated with many environmental problems created by the widespread use of pesticidal chemicals, and the chemicals often only provide a transient level of protection for crops. Chemicals also suffer from the disadvantage that all organisms in an area may be indiscriminately treated, causing needless damage to many beneficial organisms. Perhaps more importantly, many chemicals are potentially toxic to man and animals and often become concentrated in, for example, lakes and ponds and/or other water supplies.
As a result, alternate methods have been explored to reduce crop damage, one example being selective breeding of plants based upon pathogen resistance characteristics. Resistance traits, however, are sometimes controlled by many genes, making it difficult to genetically select a desired attribute to a satsfactory degree. Decreased crop yields are also occasionally encountered in resistant plants developed by selective breeding. Accordingly, there exists a strong need for compositions and methods to improve the resistance of plants from attack by pathogens. Such are provided by the present invention, which provides compositions and methods useful for genetically transforming a plant and thereby enhancing the plant""s resistance to pathogen attack.
A transgene, such as a nucleotide sequence selected in accordance with the present invention, is expressed in a transformed plant to produce in the cell a protein encoded thereby. Briefly, transcription of the DNA sequence is initiated by the binding of RNA polymerase to the DNA sequence""s promoter region. During transcription, movement of the RNA polymerase along the DNA sequence forms messenger RNA (xe2x80x9cmRNAxe2x80x9d) and, as a result, the DNA sequence is transcribed into a corresponding mRNA. This mRNA then moves to the ribosomes of the rough endoplasmic reticulum which, with transfer RNA (xe2x80x9ctRNAxe2x80x9d), translates the mRNA into the protein encoded thereby. Proteins of the present invention thus produced in a transformed host then perform an important function in the plant""s signal transduction pathway corresponding to pathogen resistance. Although the sequence of events involved in the resistance mechanism is not well understood, it is clear that proteins contemplated by the present invention enhance a plant""s resistance response by participating in this signal transduction pathway.
To comment generally upon plant resistance to pathogens, plants respond to pathogen infection in various ways, including a rapid induction of localized necrosis at the site of infection (the hypersensitive response, HR), production of antimicrobial compounds, lignin formation, oxidative burst, and increased expression of defense-related genes. Two categories of genes and, therefore, proteins are involved in a plant""s response system, disease resistance (R) genes and defense genes. R genes typically encode proteins which play a role in pathogen recognition and/or signal transduction.
R genes may be identified based upon their polymorphism in a particular plant species. That is, some crop varieties contain a particular R gene and others will lack that gene. Analysis of the progeny of genetic crosses between resistant and susceptible crop varieties allow the mapping of R genes to specific regions on a chromosome. R genes frequently, although not always, display dominant gene action and play a major qualitative role in conferring disease resistance. They frequently map to single loci in the genome and are often found to be members of a gene family. R genes differ from other genes that may play a role in disease resistance later in the defense response (after pathogen recognition). These other xe2x80x9cdownstreamxe2x80x9d genes are often referred to as xe2x80x9cdefense genesxe2x80x9d or xe2x80x9cdefense-related genesxe2x80x9d and include the class of genes known as xe2x80x9cpathogenesis-relatedxe2x80x9d (PR) genes.
With regard to increased expression of defense-related genes, it has long been recognized that transcriptional activation of a battery of plant defense-related genes is commonly associated with pathogen invasion. Defense genes include, for example, those encoding pathogenesis related proteins (PRs), hydroxyproline rich glycoproteins, and enzymes for phytoalexin biosynthesis such as phenylalanine ammonia lyase (PAL) and chalcone sythase. Although the role of these proteins in plant disease resistance is not well understood, their enzymatic functions indicate that they are well suited for defense against pathogens. Results of preliminary research have spurred extensive investigations into the biological function of defense genes and mechanisms by which they are activated.
With respect to R genes, it has been postulated that disease resistance of a plant may be induced by the genetic interaction of single genes in both the pathogen and the plant host. The phenomenon of disease resistance is believed to be initiated by physical contact between a pathogen and a potentially compatible portion of the host. Once such contact has occurred, usually as a result of wind or rain vectored deposition of the pathogen, the pathogen must recognize that such contact has been established in order to initiate the pathogenic process. Likewise, such recognition by the host is required in order to initiate a resistance response. A great deal of research is currently focused upon elucidating the precise manner in which such recognition occurs. Pathogen recognition is believed to be associated with low pH of plant tissues or the presence of plant-specific metabolites. It is believed that plant recognition occurs as a result of a race-specific mechanism where the protein product of a host disease resistance (R) gene recognizes the product of an avirulence gene of the pathogen. As a result, the plant""s defense responses are activated, leading to production of various factors (e.g., gum or cork production, production of inhibitors of pathogen proteases, deposition of lignin and hydroxyproplin-rich proteins in cell walls) and offensive resistance factors (e.g., production of phytoalexins, secreted chitinases). If the rate and level of activation of the genes producing these factors is sufficiently high, the host is able to gain an advantage on the pathogen. On the other hand, if the pathogen is fully activated at an earlier stage in the infection process, it may overwhelm both the offensive and defensive resistance factors of the plant.
In this regard, much effort has been focused on the characterization of cis-acting elements involved in elicitor- and pathogen-induced defense gene expression, and a few putative transcription factors involved in defense responses have been identified. Many defense-related genes are induced in both compatible (susceptible) and incompatible (resistant) plant-pathogen interactions. However, the expression of many defense genes is more rapid and pronounced in a plant challenged with an incompatible pathogen. In many plant-pathogen interactions, these defense responses are activated upon recognition of a pathogen carrying a specific avirulence (avr) gene by a plant host containing a corresponding R gene. In particular, incompatible interactions involving a plant R gene and a corresponding pathogen avr gene lead to accelerated plant defense gene expression. Many R genes encode proteins that are likely involved either in the recognition of signals determined by avr genes or in the early steps of signal transduction. However, a direct link between any R gene and defense gene activation has not previously been established.
In tomato, resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (which causes bacterial speck disease) has been shown to be associated with a single locus (Pto) that displays dominant gene action. Resistance of plants carrying the Pto locus to Pseudomonas syringae pv. tomato strains expressing the avirulence gene avrPto is a model system for signal transduction pathways mediated by a specific R gene. This system constitutes the only example of R gene mediated resistance pathway in which genes for multiple components have been cloned. Currently, three components are known to be involved in the signaling pathway mediated by Pto: the serine/threonine protein kinase Pto, a second serine/threonine kinase Pti1, and the leucine-rich-repeat type protein Prf. The Pto gene was originally discovered in Lycopersicon pimpinellifolium, a wild tomato species, and isolated by map-based cloning. Mutagenesis of a bacterial speck-resistant tomato line revealed a second gene, Prf, that is required for both Pto-mediated resistance and fenthion sensitivity, a related phenotype mediated by the Fen gene. Using the yeast two-hybrid system with Pto as a bait, the present inventors have identified another protein kinase Pti1 that appears to act downstream of Pto and is involved in the hypersensitive response.
In accordance with the present invention, three additional Pto-interacting proteins, Pti4, Pti5 and Pti6, also referred to herein as Pti4/5/6, that belong to a large family of plant transcription factors, are characterized. These proteins bind to a cis-element that is widely conserved among xe2x80x9cpathogenesis-relatedxe2x80x9d (PR) genes and are implicated in the regulation of these genes during incompatible plant-pathogen interactions. Pti4/5/6 each have characteristics that are typical of transcription factors. The present inventors have discovered that Pti4/5/6 specifically recognize and bind to a DNA sequence that is present in the promoter region of a large number of genes encoding PR proteins. Therefore, a direct connection has been discovered between a disease resistance gene and the specific activation of plant defense genes.
The present invention relates to the isolation, purification and use of nucleotide sequences, such as, for example, Pti4, Pti5 and Pti6 (xe2x80x9cPti4/5/6xe2x80x9d), which are useful for enhancing a plant""s ability to resist pathogen-related disease by encoding transcription factors that enhance a plant""s ability to activate defense mechanisms when faced with pathogen activity. Proteins encoded by Pti4/5/6 are useful for enhancing a plant""s ability to resist pathogen attack. The proteins encoded by the Pti4/5/6 nucleotide sequences each possess a DNA binding domain, putative nuclear localization sequences (NLS) and regions rich in acidic amino acids.
It is presently shown that the newly-isolated DNA sequences of Pti4/5/6 encode transcription factors which physically interact with Pto kinase. The present invention provides a novel form of plant protection against many types of pathogens including viruses, bacteria and fungi. While it is not intended that the present invention be limited by any mechanism whereby it achieves its advantageous result, it is believed that manipulation of these transcription factors enables the coordinate regulation of large numbers of genes involved in plant disease resistance. The invention therefore, features the DNA sequences of the Pti4/5/6 genes and the amino acid sequences of the Pti4/5/6 proteins, as set forth herein, as well as DNA sequences and amino acid sequences having substantial identity thereto and having similar levels of activity. Inventive genes may be inserted into an expression vector to produce a recombinant DNA expression system which is also an aspect of the invention.
In one aspect of the invention, inventive DNA sequences conferring disease resistance to plants are used to transform cells and to transform plants. In another aspect of the invention, there is provided a process of conferring disease resistance to plants by growing plant cells transformed with an inventive recombinant DNA expression vector and capable of expressing the DNA sequences. Plants transformed with inventive nucleotide sequences thereby have an enhanced ability to resist attack by pathogens which have an avr gene corresponding to a plant resistance gene.
It is an object of the present invention to provide isolated, sequenced and purified proteins which are useful for conferring disease resistance to a plant.
Another object of the invention is to provide isolated nucleotide sequences which encode said proteins and thereby find advantageous use when incorporated into a vector or plasmid as a transformant for a plant or microorganism.
Additionally, it is an object of the invention to provide transformed plants which have enhanced ability to resist attack by pathogens.
Further objects, advantages and features of the present invention will be apparent from the detailed description herein.